Epoxyeicosanoids Suppress Osteoclastogenesis And Prevent Ovariectomy-induced Bone Loss

Objective1. To investigate the effects of exogenous EET on the formation and function of osteoclasts.2. To obeserve the variation of the bone tissue and other variation in osteopenic model mouse after intraperitoneal injection of exogenous EET.Methods1. In vitro, both RAW264.7cells and mouse mononuclear cells obtained from bone marrow were induced by RANKL to form osteoclasts, and beisides, mCSF was added to mouse mononuclear cells.14,15-EET (cayman) effected on the RAW264.7cells and mouse mononuclear cells with the concentration of0.25μM,0.5μM,luM,2μM. The formation and function of osteoclasts were tested by methords such as staining of TRAP, activity of TRAP in the culture medium, pit formation, osteoclast-specific genes and proteins etc.2. In vivo, ovariectomy (OVX) or sham operation was performed on four month-old female C57/BL6mice,14,15-EET (0.17mg/kg) was administered to OVX mouse body by intraperitoneal injection.6weeks after operation, the activity of TRAP and inflammatory cytokines in peripheral blood were tested by TRAP ELISA kit, slices of the femur were performed staining of TRAP, and we also performed micro-computed tomography (micro-CT) of the bone histomorphometric.ResultsUsing both in vitro and in vivo studies, we observed that EETs significantly attenuated bone loss and inhibited osteoclast formation and activity, which were associated with a decreased receptor activator of NF-κB ligand (RANKL), osteoprotegerin ratio and serum levels of TNF-a and IL-1β. At the olecular level, EETs abrogated RANKL-induced activation of NF-κB, activator protein-1(AP-1), and MAPKs, including ERK and JNK, but not p38, during osteoclast formation. EETs also prevented the production of reactive oxygen species (ROS) following RANKL stimulation. As a result, EETs suppressed osteoclast-specific gene expression, including tartrate resistant acid phosphatase (TRAP), cathepsin K (CK), matrix metalloproteinase (MMP)-9, and receptor activator of NF-κB (RANK).Conclusion1. Our findings demonstrate that EETs inhibit osteoclastogenesis through modulation of multiple pathways both upstream and downstream of RANKL signaling.2. The administration or stabilized endogenous levels of EETs could represent a novel therapeutic strategy for osteoclast-related disorders, such as rheumatoid arthritis and postmenopausal osteoporosis.